Project Summary Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer. Currently, it is the fourth leading cause of cancer deaths in the United States and its incidence is rising. Over half of all patients diagnosed with PDAC have distant spread of their cancer at the time of diagnosis and the five year survival rate for these patients is only two percent. There is a critical need to understand the factors regulating PDAC liver metastasis. The proposed studies will investigate the mechanism of metastasis suppression by a novel PDAC metastasis suppressor, Inter-Alpha-Trypsin Inhibitor Heavy Chain Family Member 5 (ITIH5). Overexpressing ITIH5 in highly metastatic, low-ITIH5 PDAC cell lines suppressed cell migration, invasion and liver metastasis. In contrast, short hairpin RNA knockdown of ITIH5 in non-metastatic and ITIH5-high expressing cell lines increased migration, invasion and metastasis. Patients with ITIH5-expressing tumors lived longer than patients whose tumors did not express ITIH5. Corroborating Oncomine data show that low-ITIH5 expression is associated with shorter survival, higher tumor grade and higher tumor stage. Together these data suggest ITIH5 may inhibit PDAC progression, but the mechanism of metastasis suppression is not known. ITIH5 is a secreted serine protease inhibitor with three structural domains. However, whether secretion is required to suppress metastasis and which specific region of this protein exerts this effect are not known. Surprisingly, when we deleted the secretion signal from ITIH5, it still slowed cell motility. This opens the possibility for an intracellular role of ITIH5. Aim 1 will test the hypothesis that intracellular ITIH5 is sufficient for metastasis suppression using in vivo metastasis assays and established cell lines which express but fail to secrete ITIH5. To identify the specific region required for metastasis suppression, we will use constructs which contain individually deleted domains of ITIH5. These experiments will establish the cellular location and required domain for ITIH5-mediated metastasis suppression. Preliminary data show that ITIH5 alters transcriptional activity of several genes which characterize the cancer stem cell (CSC) phenotype in PDAC. Aim 2 will test the hypothesis that ITIH5 alters metastatic efficacy by changing the CSC phenotype. After validating identified candidates, we will use spheroid formation assays, limiting dilutions and immunohistochemistry to assess how ITIH5 changes the CSC phenotype in PDAC. Finally, in vivo metastasis assays using dissociated spheroids will define how ITIH5 changes the CSC phenotype and alters metastasis. In conclusion, the proposed studies investigate the mechanism of a new metastasis suppressor gene ITIH5 in orthotopic mouse models of metastasis. Together, these data will define the cellular location and specific region required for ITIH5-mediated metastatic suppression and the molecular mediators of this signaling in PDAC. These data will enrich our understanding of PDAC metastasis and may ultimately help improve the prognosis of patients with metastatic pancreatic cancer.